Cholera is an acute water-borne diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139. The bacterium is transmitted person-to-person by the fecal oral route. Infecting Vibrios that overcome the gastric acid barrier swim toward and adhere to the intestinal mucosa and express major virulence factors such as the toxin co-regulated pilus and cholera toxin, which is largely responsible for the profuse rice-watery diarrhea typical of this disease. At a late stage of infection, V. cholerae down-regulates the expression of virulence factors and detaches to return to the environment. Previous studies in our laboratory showed that the RpoS- dependent Zn-metalloprotease hemagglutinin (HA)/protease encoded by hapA promote detachment of V. cholerae from cultured cells and mucus. A study conducted in the rabbit ileal loop model subsequently demonstrated that V. cholerae detachment or mucosal escape is facilitated by RpoS which enhances the expression of motility. It has been recently shown that freshly shed Vibrios in cholera stool are in a hyper infective state that could promote person-to-person cholera transmission. Therefore, detachment of hyper infective Vibrios from the intestinal mucosa is a key step in the cholera transmission chain. We have studied the regulatory circuitry that controls the expression of HA/protease. These studies have unveiled a complex regulatory network involving the cyclic AMP receptor protein (CRP), the master quorum sensing regulator HapR, the histone-like nucleoid structuring protein (H-NS), and the general stress response regulator RpoS that act in concert to inhibit the expression of virulence and to enhance the production of HA/protease and motility. The central hypothesis of this research plan is that the coordinated up-regulation of HA/protease and motility late in infection promotes V. cholerae detachment or mucosal escape in a synergistic manner. In this study we propose to rigorously test this hypothesis by integrating a variety of approaches such as bacterial genetics, chromatin immunoprecipitation (ChIP), ChiP-sequencing, confocal microscopy and animal models. In Aim 1, we will determine the regulatory interactions that trigger V. cholerae detachment late in infection. To this end, we plan to examine how H-NS and RpoS regulate the expression of RpoN and FlrA to coordinate the expression of flagellar motility with HA/protease production. We will use ChIP to determine if RpoS (CS) directly participates in transcription of rpoN, flrA and hapA. In addition, immunoprecipitates will shipped to GenPathway (San Diego, CA) to determine the RpoS binding sites in the V. cholerae genome. This data will be used to putatively identify intermediate regulators linking RpoS with the regulation of motility, HA/protease and/or promoting detachment. In Aim 2, we will examine the role of motility and HA/protease in detachment in vivo. To this end, we will use the rabbit ileal loop model, confocal microscopy and fluorescently-labeled isogenic mutants to define the contribution HA/protease and motility to mucosal escape. In addition, we will investigate if RpoS enhances the expression of additional detachment factors by comparing mucosal escape in RpoS-positive and negative isogenic strains that lack flaA (non-motile, non-flagellated) and hapA. Finally, we will use an infant mouse hyper infectivity assay to examine the temporal relationship between induction of hyper infectivity and detachment. We expect that a better understanding of the events that inhibit virulence and promote detachment late in infection will open new avenues to treat cholera and provide important clues to explain the epidemic potential of this human pathogen. PUBLIC HEALTH RELEVANCE: Cholera continues to cause seasonal outbreaks in highly populated regions in Asia, Africa and Latin America. This disease is caused by Vibrio cholerae of serogroups O1 and O139 which are transmitted person-to-person by the fecal oral route. V. cholerae colonizes the human small intestine, secretes cholera toxin to cause diarrhea and detaches late in infection to return to the aquatic environment. Freshly shed (detached) Vibrios are in a "hyper infective" physiological stage that could enhance person-to person transmission and cholera spread. Therefore, detachment of hyper infective bacteria from the intestinal mucosa late in infection is a critical step of the cholera transmission chain. The general aim of our research is to establish the regulatory interactions that trigger V. cholerae detachment and the role of protease production and motility in this process. These studies could open new avenues to treat cholera and shed light on the epidemic potential of this pathogen.